Influenza: A Century of Research

By Irina Kiseleva and Natalie Larionova

Influenza is one of the most ancient and intriguing diseases that has been accompanying our civilization for millennia. While mankind has successfully defeated many dangerous influenza infections in the last couple of centuries, influenza control remains a serious problem for public health. A number of influenza vaccines and antiviral compounds have been licensed in recent times. However, the influenza virus is still ahead of us, as it continues to persistently infect humans to this day. Influenza: A Century of Research shows how influenza virology has developed historically and the tremendous knowledge that has been uncovered in the study of influenza. In this monograph, the authors present a historical perspective on influenza, chronologically, with an emphasis on its virology. Chapters cover information about the isolation of the first influenza viruses, substrates, and models for studying influenza, structure, and life cycle of the influenza virus, mechanisms of attenuation and virulence. Chapters progress into the multidisciplinary aspects of influenza research such as influenza virus ecology and the evolutionary origin of epidemic and pandemic influenza viruses. A significant part of the book also covers the description of the prevention and treatment of influenza and reasons that have contributed to insufficient control for influenza. The questions of how the COVID-19 pandemic affects the circulation of seasonal respiratory viruses, and if we can eliminate this virus are also addressed.

Influenza: A Century of Research is an informative source of information for a broad range of readers, academic or otherwise, who are interested in knowing more about the disease.

• Language: English
• Publisher: Bentham Science Publishers
• Release date: Jul 7, 2021
• ISBN: 9781681088440

Book preview

PREFACE

Influenza is one of the most mysterious and ancient diseases that have been accompanying our civilization for centuries. The first written description of influenza-like disease belongs to Hippocrates (5th century BC). However, the exact age of the influenza virus is unknown.

Humankind has successfully defeated many dangerous infections that caused harm to human health and safety. Plaque, polio, smallpox, tuberculosis, measles, and many other infectious diseases were completely eradicated or controlled. In contrast, influenza control remains a serious problem for public health.

According to the World Health Organization, influenza is responsible for 3-5 million severe illnesses worldwide and up to 650 thousand respiratory deaths. The unpredictable character of the influenza virus spread is a significant threat to humans. In the last eight decades after the first influenza virus was isolated, huge progress has been made in studying, preventing, and treating influenza. Several influenza vaccines and antiviral compounds have been licensed. However, the influenza virus is still ahead of us. Therefore, this eBook aims to show how influenza virology has developed historically. We briefly demonstrate the tremendous success that has been achieved in the study of influenza over the past 100 years and discuss if there is a way to control this infection.

A lot has been written about influenza already. PubMed and other online resources supporting the search and retrieval of peer-reviewed biomedical and life sciences literature comprise over 200,000 articles, reviews, and books. In this eBook, we have tried to illuminate knowledge on influenza from a historical perspective, chronologically, with an emphasis on the virological part of the studies.

Chapter 1 describes a history of isolation of the first influenza viruses and dwells on the historical aspect of the development of appropriate substrates and models for studying influenza. Chapter 2 addresses the genome and capsid structure, molecular mechanisms of replication, and life cycle of the influenza virus. Mechanisms involved in the attenuation and virulence of the influenza virus are also discussed. Chapter 3 presents a comprehensive review of the influenza virus ecology and evolution. The origin of epidemic and pandemic influenza viruses is discussed as well. Chapter 4 focuses on influenza prophylaxis and treatment. Historical aspects of current achievements in this field were reviewed. Reasons contributed to insufficient control for influenza are highlighted in Chapter 5. The main properties of the influenza virus that may influence control for influenza are described. The COVID-19 pandemic affected both all aspects of our lives and the circulation of seasonal respiratory viruses. Chapter 6 describes some issues arising with the spread of pandemic viruses in general and SARS-CoV-2 infection in particular.

The short final Chapter 7 has been written in the form of conclusion; it is devoted to briefly summarize the 100-year study of influenza. Despite the huge number of research and years or rather centuries of research, a cure for the virus has not yet been found. The possibility of defeating influenza in the nearest future is discussed.

The intended audience for this eBook includes students of biological and medical colleges, Ph.D. students, post-docs, a wide range of virologists who are specialized in the field of influenza, and everyone interested in this infection.

CONSENT FOR PUBLICATION

Not applicable.

CONFLICT OF INTEREST

The author declares no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

Finally, the authors would like to thank Dr. Vladimir Zarubaev for writing the foreword, Bentham Science Publishers for the continuous support throughout the process of writing this book, and the Russian Science Foundation (grant 21-75-30003) for the financial support.

Irina Kiseleva

& Natalie Larionova

Institute of Experimental Medicine

St. Petersburg

Russia

Historical Aspects

Irina Kiseleva, Natalie Larionova

Abstract

The first animal influenza A virus was isolated in 1931 by Richard Shope. The virus caused a highly contagious, influenza-like disease in pigs. Two years later, in 1933, the first human influenza A virus was isolated by Wilson Smith and colleagues. Soon after, in 1940, a representative of influenza virus type B was discovered by Thomas Francis, Jr. Being obligate intracellular parasites, viruses can be cultivated only within sensitive substrates. Three main substrates for the cultivation of influenza viruses are known: sensitive animals, embryonated chicken eggs, and tissue cultures. Today, in the twenties of the 21st century, sensitive animals are not often used for the isolation of the infectious virus. However, they are widely used to study and model a number of infectious diseases, including influenza. A list of these animals used for influenza research is very long, starting from ferrets and mice and ended with exotic zebrafish.

Keywords: Animal models, Embryonated chicken eggs, First human influenza viruses, Influenza, Nomenclature, Tissue culture.

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INTRODUCTION

Influenza is one of the most mysterious and ancient diseases known for centuries. The exact age of the influenza virus is unknown. Humankind has successfully eradicated many deadly infections like plaque, polio, etc. While acknowledging the measures of the World Health Organization (WHO), Centers for Disease Control and Prevention (CDC), and other specialized agencies responsible for international and national public health, have taken to control influenza, it remains a serious cross-border problem. According to WHO, influenza is responsible for million cases of severe illnesses worldwide and up to 650 thousand respiratory deaths [1]. The unpredictable character of the influenza virus spread is a significant threat to humans.

Isolation of the first influenza viruses resulted in a rapid flowering of influenza research. The right choice of adequate animal models and substrates for virus cultivation is very important. Therefore, in this chapter, the historical aspect of the development of appropriate substrates and models for studying influenza is discussed.

THE FIRST INFLUENZA VIRUSES

Influenza viruses belong to the Orthomyxoviridae family. Members of this family, which include seven genera [2, 3], are characterized by similarities in the structure of the viral particle and the way of reproduction. These are enveloped RNA viruses with a single-stranded segmented minus-stranded (negative-stranded) genome [4, 5]. Looking back in history, it is remarkable that the first influenza virus was isolated in Italy 119 years ago but classified as fowl plaque or fowl pox virus. Fifty years later, it was found that this is one of the highly pathogenic avian influenza A viruses.

In 1931, Richard Shope isolated a filterable agent that caused a highly contagious, influenza-like disease in pigs [6-8]. Soon after, during the 1933 epidemic, the first human influenza A virus was isolated by Smith et al. [9]. After several unsuccessful attempts to infect different species such as guinea pigs, mice, snakes, and hedgehogs, it was found that only ferrets exhibited catarrhal, nasal, and temperature symptoms of respiratory disease similar to humans. In 1940, the first human influenza B virus was discovered [10], and in 1968, the new influenza A virus subtype H3N2 was identified in tissue culture [11]. In 1947, in the United States, type C influenza virus C/Taylor/1233/1947 was identified in a human with upper respiratory symptoms [12].

In the early 1950s, the type D influenza virus (Sendai strain) that typically affects rodents and is not pathogenic for humans was isolated. However, later it was discovered that the Sendai virus belongs to the family Paramyxoviridae. The real influenza D virus belongs to the recently characterized new genus in the Orthomyxoviridae family. This virus was originally detected in pigs in the US [13]; however, cattle are now believed to be the main reservoir of the type D influenza viruses [13-15].

The taxonomic division of influenza viruses is based on their antigenic characteristics. According to the antigenic specificity of the internal structural components of the virion: the nucleoprotein (NP) and the matrix protein (M1), they are subdivided into four genera A, B, C and D [2, 3]. Type C and D have 7 RNA segments and encode 9 proteins, while types A and B have 8 RNA segments. Viruses belonging to different genera do not have common antigens, differ in epidemiological features and the severity of the disease they cause. Genetic reassortment between viruses of different genera does not occur. Comparative characteristics of influenza viruses A, B, C and D are presented in Table 1.

Table 1 Types and subtypes of influenza viruses.

¹Not applicable.

Influenza A virus causes the most severe diseases and may infect a wide range of host species. There are 18 hemagglutinin (HA) subtypes and 11 neuraminidase (NA) subtypes, respectively [16]. Among 198 potential influenza A subtype combinations, 131 subtypes have been detected in wild nature [30]. Serotypes of HA, in turn, form two phylogenetic groups that differ significantly from each other, but within each group, the domain encoding the HA stalk is antigenically similar: group 1 includes hemagglutinins H1, H2, H5, H6, H8, H9, H11 – H13, H16 and H17, group 2 - hemagglutinins H3, H4, H7, H10, H14 and H15 [31]. Two subtypes of influenza A viruses are routinely circulating in humans: A (H1N1)pdm09 and A (H3N2). Influenza virus ecology and evolution will be discussed in detail in Chapter 3.

Human influenza A viruses cause annual epidemics and occasionally pandemics. In addition, viruses of zoonotic origin sporadically infect humans, causing severe respiratory infections and high mortality. Most zoonotic viruses are incapable of sustained human-to-human transmission, but mutations or reassortment with human influenza viruses in extremely rare cases leads to the emergence of a new virus with pandemic potential that can be transmitted by airborne droplets [32]. Only three serotypes of HA (H1, H2, and H3) and two serotypes of NA (N1 and N2) were integral parts of the pandemics pathogens and circulated widely among humans during the era of influenza studies [33]. The annual (seasonal) epidemics due to influenza A viruses are caused by the high rate of antigenic mutations that allow the virus to escape the immune defenses. Influenza viruses of genera B and C are not subdivided into serotypes, and no pandemics occur with their participation [34].

Influenza B viruses cause seasonal epidemics with the typical pattern of influenza infection. They are subject to antigenic variability too, although the rate of appearance mutations is 3-5 times lower compared to influenza A viruses [34, 35]. Since the early 1980s, two branches (lines) of the influenza B virus have been circulating alternately and jointly, differing significantly in antigenic characteristics of HA and NA and other properties [19, 35]. Influenza B virus causes less severe diseases than influenza A virus but can cause outbreaks and the influenza C virus causes acute respiratory illness most commonly in infants and young children, usually only associated with mild upper respiratory illness.

Influenza D virus has been identified recently. Its primary reservoir is cattle. Sheep, goats, pigs, horses, and camels are also susceptible to infection [3, 17, 36]. It is still unknown whether the influenza D virus causes disease in humans, but 97% of people in contact with cattle in Florida have antibodies to the influenza D virus [29].

MODELS FOR STUDYING INFLUENZA

Laboratory animals are widely used to study and model a number of diseases of an infectious and non-infectious nature, as well as for isolation of infectious agents. Animals, especially vertebrates, have repeatedly been used throughout the history of medical and biological discoveries and to this day, play a key role in biomedical researches. The first references to animal studies could be found in the writings of Greek philosophers and physicians Aristotle and Erasistratus, who most likely were the first to perform experiments on living animals. Louis Pasteur was the pioneer in the use of animals to prove the infectious nature of some diseases, such as anthrax [37, 38] or rabies [39].

Fig. (1))

Use of laboratory animals, %. The areas of animal models’ use are wide.

Since the 18th century, the use of laboratory animals in experimentation became more common and the numbers of laboratory animal procedures conducted continue to rise progressively [40-42]. The areas of animal models’ use can be seen in Fig. (1). The majority of animals are used in biomedical research (testing vaccines and biologicals, cancer research, heart diseases, and circulation research, etc.), basic research including military, space, etc., drug research (toxicity tests, cosmetics, new antiviral compounds, etc.) and in education.

Animal research is now becoming even more prevalent - it is estimated that the number of mice and rats used in research has been increasing every year. Over 50 years ago, the principles of the 3Rs (Replacement, Reduction, and Refinement) providing a framework for performing more humane animal research were developed (Fig. 2). 3Rs are guiding principles for the more ethical use of animals in preclinical studies. Replacement methods which avoid/replace the use of animals; reduction methods which minimize the number of animals used per experiment; refinement methods which minimize animal suffering and improve welfare. The questions of how to refine to lessen pain, reduce the number of animals used, and replace with non-animal methods and how many animals should be included in the group are under discussion [43, 44].

Fig. (2))

Three guiding principles for the more ethical use of animals in preclinical studies (3Rs principles).

Ferrets were the first animals successfully used for the isolation of the first human influenza virus (see section The first influenza viruses above). Currently, ferrets are widely used to study the pathogenesis of influenza infection (Fig. 3) [45-51] and transmissibility of influenza viruses [45, 47, 50]. Moreover, according to WHO recommendations [52], ferrets are used for preclinical characterization of potentially pandemic influenza vaccines [53-60].

Fig. (3))

Nasal symptoms and gross examination of the lungs of ferrets infected with A/South Africa/3626/2013 (H1N1)pdm09 influenza virus [61]. (a, b) The ferrets were inoculated with the virus. (a) Severe lung lesions. (b) Nasal discharge on the external nares (red arrow). (c) The lungs of the ferret inoculated with phosphate-buffered saline.

A wide range of other experimental animals (mice, poultry, guinea pigs, cotton rats, pigs, hamsters, macaques) are used to study the various aspects of the manifestation of influenza infection and the selection of therapeutic and prophylactic drugs to treat influenza infection (Table 2) [47, 50, 62]. The most common models are mice (over 70% of all models used). A zebrafish has been actively used in the past few years to study the immune response to influenza and the selection of anti-influenza chemotherapy drugs [63-65]. Another exotic animal, the tree shrew, was shown to be physiologically and genetically related to primates, which make it a potential animal model of human diseases [66, 67]. Proper modeling of various aspects of the manifestation of influenza infection in the relevant sensitive animals is the key to scientific success.

Table 2 Animal models in influenza research.

The difference between a sensitive animal model as a substrate for virus isolation or research and a natural reservoir (host) of the influenza virus should be clearly understood (Fig. 4). Mice, ferrets, guinea pigs, etc., are very sensitive experimental animal models, but they never meet the influenza virus in their natural habitats. Between animals-substrates and animals-hosts, an additional group may be distinguished – animals who are not natural hosts but maybe occasionally infected. An accidental host accidentally harbors an organism that is not ordinarily parasitic in the particular species and usually does not infect it. Influenza A viruses can infect species other than the natural hosts in which they normally circulate on rare occasions.

Fig. (4))

Natural reservoirs and laboratory models for influenza A viruses.

For instance, humans could become accidental hosts for the avian influenza A (H5N1) virus. While avian influenza A viruses are highly species-specific, they may occasionally cross the species barrier to infect other species causing a disease of high lethality (Fig. 5).

Fig. (5))

Laboratory-confirmed human cases of avian influenza A (H5N1) reported to WHO in 2003-2020 and mortality rate (reprinted with permission from Book Publisher International) [98]. ¹The number of deaths (%) from the total number of laboratory-confirmed H5N1 cases. ²Data from January 2020 through 28 February 2020.

This disease should not be confused with seasonal human influenza, generally caused by human H1N1pdm09, H3N2, or B viruses. Episodic transmission of avian influenza viruses to humans occurs when there is close contact with infected birds. Due to the high lethality and virulence, the avian influenza virus of subtype H5N1 is one of the world's largest pandemic threats.

Ferrets being a highly experimental model that is sensitive to the influenza virus, could occasionally become the accidental hosts. During the influenza season, laboratory breeding ferrets can catch influenza from sick lab technicians or ferret's farmworkers. Influenza disease in ferrets can be fatal (Fig. 3).

For obtaining adequate results and their correct assessment, three important conditions must be met:

The right choice of anesthetic for animal study.

Standardization of the infectious dose.

The right choice of the virus as a model for research.

The Right Choice of Anesthetic for Animal Study

Virological and histopathological examinations occupy an important role in preclinical animal studies of influenza. A whole range of manipulations with animals is carried out under anesthesia to minimize their suffering. The refinement of anesthetic choice is an important part of experimental procedures. Ketamine is widely used in veterinary practice as a surgical anesthetic for general anesthesia. However, in some countries, ketamine is included in the list of narcotic drugs, psychotropic substances, and their precursors, which are subject to control, and its use is strictly limited. In veterinary practice, isoflurane is also broadly used as an anesthetic agent. Efficacy and safety of isoflurane have been shown in a number of nonclinical [99, 100] and clinical [101] trials. However, isoflurane may induce airway irritation [102] and should be used with caution in studies of the pathogenesis of respiratory viruses. It was shown that a five-fold inhalation of isoflurane might dramatically affect lung tissue and cause injuries in the form of hemorrhagic lung edema. Gross necropsy examination revealed gross lung lesions in a mock-inoculated group similar to animals infected with wild-type (WT) influenza virus [103]. This finding corresponded to a previous study [104]. The authors reported that general anesthesia with volatile agents, including isoflurane, provoked lung injury, acute inflammatory response, and leukocytic infiltration in rats. In contrast, the absence of any non-specific lung damages in ferrets caused by the injection of zoletil 100 was reported in studies of influenza virus [103, 105, 106] (Fig. 6).

Fig. (6))

Macroscopic view of the lungs of ferrets after multiple rounds of anesthesia (reprinted with permission from Vaccine Research) [103]. Yellow arrows – pulmonary hemorrhages. Ferrets received five applications of anesthesia. Isoflurane maintained inhalation anesthesia; intramuscular anesthesia was induced by injection with zoletil 100. At the end of the experiment, ferrets of all groups were humanely euthanized with a combination of Zoletil 100 and xylazine. (a) The ferret was anesthetized with isoflurane and received only PBS (placebo); (b) The ferret was anesthetized with isoflurane and inoculated with WT influenza B virus; (c) The ferret was anesthetized with zoletil 100 and received only PBS (placebo); (d) The ferret were anesthetized with zoletil 100 and inoculated with WT influenza B virus.

Standardization of the Infectious Dose

Standardization of the infectious dose is very important for study design and subsequent interpretation of the results. The literature describes three options for standardizing the dose of influenza virus to be used for infect experimental animals: (i) the 50% minimum infectious dose (MID50) [107, 108]. Depending on the objectives, animals were